Plasmodium falciparum infections cause malaria resulting in the deaths of ~435,000 people, mostly children, in malaria-endemic regions each year. A distinct feature of P. falciparum is sequestration; the adhesion of infected RBCs (iRBCs) containing mature parasites to endothelial cells. iRBCs display surface bumps known as knobs that mediate the attachment of iRBCs to endothelium. Both parasite and host proteins located at the knobs contribute to the cytoadhesion phenotype. Strategies that can disrupt interactions of knobs offer potential therapeutic means of attenuating their adhesive properties; however, their precise mechanisms remain poorly understood. Using phage display cDNA screens, we identified a new knob parasite protein termed PfGARP. P. falciparum glutamic acid-rich protein (PfGARP) is exported outside of the RBCs and binds to an extracellular segment of band 3. This novel localization of PfGARP suggests a functional role in the adhesion phenotype of iRBCs. Based on these findings, we hypothesize that PfGARP contributes to the cytoadhesion of malaria infected human RBCs to vascular endothelium in a PfEMP1-independent manner by engaging a novel ligand-receptor(s) complex on the endothelial surface. We will test this hypothesis as follows: Specific Aim 1: Mechanism of PfGARP interactions with human erythrocytes/RBCs. Experimental support comes from our immuno-colocalization studies with known knob markers, PfEMP1 and KAHRP, placing PfGARP at the knobs. Since a truncated form of band 3, which binds to PfGARP, is also localized to knobs, this raises the possibility that the PfGARP-Band 3 complex may function as an PfEMP1-independent interaction linking iRBCs to the endothelial cells. We propose to identify the precise ectoplasmic segment of band 3 and critical amino acid residues that mediate Band 3-PfGARP interaction. We plan to use the high-affinity peptides of band 3 for assessing the functional effects of PfGARP binding to human RBCs including adhesion, deformability, and band 3 phosphorylation. We will employ immuno-EM, immunoprecipitation and mass spectrometry to investigate the localization and composition of PfGARP complex on knobs. A PfGARP knockout parasite strain will be generated to evaluate the adhesive properties of iRBCs by multiple cytoadhesion assays. Aim 2: Identification of host endothelial receptors for PfGARP. We generated a phage display cDNA library from human endothelial cells (TNF?-activated HUVECs) and screened with recombinant PfGARP as bait. Among several potential receptors identified in the screens, we are currently characterizing a single-pass transmembrane receptor termed CLEC14A (C-type lectin domain containing 14A) as a potential binding partner of PfGARP. To test the possibility that PfGARP engages CLEC14A to recruit iRBCs to the vascular endothelium, we will use state-of-the-art imaging tools developed for endothelial cell lines in the Herman laboratory at Tufts to monitor real-time kinetics of cytoadherence ex vivo. Together, the proposed studies will contribute to our understanding of the biological function of knobs and role of PfGARP in the adhesion of iRBCs to microvascular endothelial cells.